Secondary structure in the solution conformation of the proteinase inhibitor IIA from bull seminal plasma by nuclear magnetic resonance

Nuclear magnetic resonance data on the protease inhibitor IIA from bull seminal plasma were used to determine the secondary structure elements in the solution conformation of the protein. The experimental data were obtained from analyses of two-dimensional 1H nuclear magnetic resonance spectra at 500 and 360 MHz and include details of inter-residue nuclear Overhauser enhancements, vicinal spin-spin coupling constants and the sequence location of slowly exchanging amide protons. Accurate measurement of coupling constants and reliable assignments of nuclear Overhauser enhancements were facilitated by the use of absorption mode two-dimensional spectroscopy and large data matrices. It is shown that the peptide backbone is extended from residues 4 to 7, followed by a poorly defined helical region from residues 8 to 13 with a marked change of direction at residue Phe10. Residues 15 to 19 are extended and there is a kink at residue Glu20. Residues 22 to 27 form the central strand of a triple-stranded antiparallel beta-sheet, of which the other two strands are residues 29 to 33 and 49 to 53. Residues 34 to 46 form a helix. The tight turn in the beta-sheet is of type I geometry, and there is a beta-bulge at residue His53.     

Systematic application of two-dimensional 1H nuclear-magnetic-resonance techniques for studies of proteins. 2. Combined use of correlated spectroscopy and nuclear Overhauser spectroscopy for sequential assignments of backbone resonances and elucidation of polypeptide secondary structures

This paper describes a new nuclear magnetic resonance approach for the determination of secondary structure in globular proteins. To illustrate the practical application of the new procedure, two-dimensional correlated spectroscopy and two-dimensional nuclear Overhauser enhancement spectroscopy were used to obtain individual assignments for all the backbone protons of the beta-sheet secondary structures in the basic pancreatic trypsin inhibitor. First, combined connectivity diagrams of these two methods recorded in both 2H2O solution and H2O solution of the inhibitor were employed to obtain sequential, individual resonance assignments for the separate strands in the beta sheet. Second, a 2D nuclear Overhauser enhancement spectrum recorded with a long mixing time was used to determine how the separate, extended polypeptide strands are linked by hydrogen bonds in the sheet structures. By combination of these results with the identifications of the amino acid side-chain resonances described in the preceding paper, the beta-sheet structures can, without reference to data on the spatial structure obtained with other techniques, be localized in the amino acid sequence. This investigation confirms results on limited regions of the beta sheet in the inhibitor obtained previously with one-dimensional nuclear magnetic resonance experiments and demonstrates that the entire beta-sheet structure seen in single crystals of the inhibitor is preserved in aqueous solution.     

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.     

Proton NMR assignments and secondary structure of the snake venom protein echistatin.

The snake venom protein echistatin is a potent inhibitor of platelet aggregation. The inhibitory properties of echistatin have been attributed to the Arg-Gly-Asp sequence at residues 24-26. In this paper, sequence-specific nuclear magnetic resonance assignments are presented for the proton resonances of echistatin in water. The single-chain protein contains 49 amino acids and 4 cystine bridges. All of the backbone amide, C alpha H, and side-chain resonances, except for the eta-NH of the arginines, have been assigned. The secondary structure of the protein was characterized from the pattern of nuclear Overhauser enhancements, from the identification of slowly exchanging amide protons, from 3JC alpha H-NH coupling constants, and from circular dichroism studies. The data suggest that the secondary structure consists of a type I beta-turn, a short beta-hairpin, and a short, irregular, antiparallel beta-sheet and that the Arg-Gly-Asp sequence is in a flexible loop connecting two strands of the distorted antiparallel beta-sheet.     

A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult hemoglobin

High-resolution proton nuclear magnetic resonance spectroscopy and nuclear Overhauser effects for the low-field exchangeable proton resonances of human normal adult hemoglobin in aqueous solvents are being used to confirm and extend the assignments of these resonances to specific protons at the intersubunit interfaces of the molecule. Most of these exchangeable proton resonances of human normal adult hemoglobin have been found to be absent in the spectra of isolated alpha or beta subunits. This finding indicates that they are specific spectral markers for the quaternary structure of the hemoglobin tetramer. Based on the nuclear Overhauser effect results, we have assigned the exchangeable proton resonance at +7.4 ppm downfield from H2O to the hydrogen-bonded proton between alpha 103(G10)His and beta 108(G10)Asn at the alpha 1 beta 1 interface. The nuclear Overhauser effect results have also confirmed the assignments of the exchangeable proton resonances at +9.4 and +8.2 ppm downfield from H2O previously proposed by workers in this laboratory based on a comparison of human normal adult hemoglobin and appropriate mutant hemoglobins. This independent confirmation of previously proposed assignments is necessary in view of the possible long-range conformational effects of single amino-acid substitutions in mutant hemoglobin molecules.     

Assignment of paramagnetically shifted resonances in the 1H NMR spectrum of horse ferricytochrome c.

The proton resonances of the heme, the axial ligands, and other hyperfine-shifted resonances in the 1H nuclear magnetic resonance spectrum of horse ferricytochrome c have been investigated by means of one- and two-dimensional nuclear Overhauser and magnetization transfer methods. Conditions for saturation transfer experiments in mixtures of ferro- and ferricytochrome c were optimized for the cross assignment of corresponding resonances in the two oxidation states. New resonance assignments were obtained for the methine protons of both thioether bridges, the beta and gamma meso protons, the propionate six heme substituent, the N pi H of His-18, and the Tyr-67 OH. In addition, several recently reported assignments were confirmed. All of the resolved hyperfine-shifted resonances in the spectrum of ferricytochrome c are now identified. The Fermi contact shifts experienced by the heme and ligand protons are discussed.     

NMR studies of the trp repressor from Escherichia coli. Characterisation and assignments of residue types

High-resolution proton nuclear magnetic resonance spectra of the trp repressor of Escherichia coli under various conditions are reported and analysed. The spectrum of the denatured state agrees with that predicted from the amino acid composition, with the exception of the two histidine residues, which have different chemical shifts although they titrate normally. The spectrum of the native protein shows the presence of extensive secondary and tertiary structure. Using information from chemical shifts, numbers of protons, titration behaviour, homonuclear chemical-shift-correlated spectroscopy and nuclear Overhauser enhancement correlated spectroscopy, most of the aromatic protons have been assigned to residue type. Further, about 30% of the aliphatic protons have been assigned to residue type by two-dimensional spectroscopy. Nuclear Overhauser enhancements establish that high-field methyl groups belonging to a valine residue lie directly over an aromatic ring.     

Assignment of the proton NMR spectrum of reduced and oxidized thioredoxin: sequence-specific assignments, secondary structure, and global fold

Complete proton assignments are reported for the 1H nuclear magnetic resonance (NMR) spectrum of Escherichia coli thioredoxin in the oxidized (with active-site disulfide bridge) and reduced (with two sulfhydryl groups) states. The assignments were obtained by using an integrated assignment strategy in which spin systems were identified from a combination of relayed and multiple quantum NMR techniques prior to sequential assignment. Elements of secondary structure were identified in each protein from characteristic nuclear Overhauser effects (NOE), coupling constants, and slowly exchanging amide protons. In both oxidized and reduced thioredoxin, approximately 33% of the 108 amino acid residues participate in a beta-sheet containing four major strands (three antiparallel and one parallel). A further short beta-strand is connected in a parallel fashion at the N-terminal end of the sheet. Two of the antiparallel beta-strands are connected by a 7-residue beta-bulge loop. Three helical segments, also containing approximately 33% of the amino acid residues, are well-defined in both oxidized and reduced thioredoxin. The remaining third of the molecule apparently consists of reverse turns and loops with little defined secondary structure. The global folds of oxidized and reduced thioredoxin are shown to be essentially identical. Both NOE connectivities and chemical shift values for the two proteins are very similar, except in the immediate vicinity of the active site where significant variations in the chemical shift indicate subtle conformational changes. While the overall fold of oxidized thioredoxin is the same in solution and in the crystalline state, some small differences in local conformation are apparent.     

Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of human [113Cd7]metallothionein-2 was determined by nuclear magnetic resonance spectroscopy in solution. Sequence-specific 1H resonance assignments were obtained using the sequential assignment method. The input for the structure calculations consisted of the metal-cysteine co-ordinative bonds identified with heteronuclear correlation spectroscopy, 1H-1H distance constraints from nuclear Overhauser enhancement spectroscopy, and spin-spin coupling constants 3JHN alpha and 3J alpha beta. The molecule consists of two domains, the beta-domain including amino acid residues 1 to 30 and three metal ions, and the alpha-domain including residues 31 to 61 and four metal ions. The nuclear magnetic resonance data present no evidence for a preferred relative orientation of the two domains. The polypeptide-to-metal co-ordinative bonds in human metallothionein-2 are identical to those in the previously determined solution structures of rat metallothionein-2 and rabbit metallothionein-2a, and the polypeptide conformations in the three proteins are also closely similar.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Basic pancreatic trypsin inhibitor

The assignment of the ¹H nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor with the use of two-dimensional ¹H nuclear magnetic resonance techniques at 500 MHz is described. The assignments are based entirely on the known amino acid sequence and the nuclear magnetic resonance data. Individual resonance assignments were obtained for all backbone and C^β protons, with the exception of those of Arg1, Pro2, Pro13 and the amide proton of Gly37. The side-chain resonance assignments are complete, with the exception of Pro2 and Pro13, the N^δ protons of Asn44 and the peripheral protons of the lysine residues and all but two of the arginine residues.     

Sequential resonance assignment and secondary structure determination of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors

The solution conformation of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors, has been investigated by nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments of the 1H NMR spectrum have been obtained by using a number of two-dimensional techniques for identifying through-bond and through-space (less than 5-A) connectivities. Elements of regular secondary structure have been identified on the basis of a qualitative interpretation of the nuclear Overhauser enhancement, coupling constant, and amide exchange data. These are two beta-sheet regions. One double-stranded antiparallel beta-sheet comprises residues 11-14 (strand 1) and 37-39 (strand 2). The other triple-stranded sheet is formed by two antiparallel strands comprising residues 45-49 (strand 4) and 53-57 (strand 5) connected by a turn (residues 50-52), and a small strand consisting of residues 20-22 (strand 3) that is parallel to strand 4.     

Individual assignments of amide proton resonances in the proton NMR spectrum of the basic pancreatic trypsin inhibitor.

Studies of proton-proton nuclear Overhauser effects were used to obtain individual assignments of 17 amide proton resonances in the 360 MHz proton nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor. First, optimizing the conditions for obtaining selective nuclear Overhauser effects in the presence of spin diffusion in macromolecules is discussed. Truncated driven nuclear Overhauser experiments were used to assing the amide proton resonances of the beta-sheet in the inhibitor. It is suggested that these techniques could serve quite generally to obtain individual resonance assignments in beta-sheet secondary structures of proteins. Combination of nuclear Overhauser studies with spin decoupling further resulted in individual assignments of the gamma-methyl resonances of the two isoleucines and numerous Calpha and Cbeta protons.     

Sequence-specific 1H NMR assignment and secondary structure of the Arc repressor of bacteriophage P22, as determined by two-dimensional 1H NMR spectroscopy

The Arc repressor of bacteriophage P22 is a DNA binding protein that does not belong to any of the known classes of such proteins. We have undertaken a 1H NMR study of the protein with the aim of elucidating its three-dimensional structure in solution and its mode of binding of operator DNA. Here we present the 1H nuclear magnetic resonance (NMR) assignments of all backbone protons and most of the side-chain protons of Arc repressor. Elements of secondary structure have been identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser enhancements (NOEs). Two alpha-helical regions have been found in the peptide regions 16-29 and 35-45. The ends of the helices could not yet be firmly established and could extend to residue 31 for the first helix and to residue 49 for the second. Immediately before the first helix, between residues 8 and 14, a region is present with beta-sheet characteristics dominated by a close proximity of the alpha-protons of residues 9 and 13. Because of the dimeric nature of the protein there are still two possible ways in which the NOEs in the beta-sheet region can be interpreted. If the NOEs are intramonomer, this requires a tight turn involving residues 10-12. Alternatively, if the NOEs are intermonomer, then and antiparallel beta-sheet would be implicated comprising two strands of different Arc monomers. While the data presently do not allow an unambiguous choice between these two possibilities, some evidence is discussed that favors the latter (beta-sheet between monomers).(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional NMR studies of the antimicrobial peptide NP-5

Nearly complete proton resonance assignment of the rabbit antimicrobial peptide NP-5 has been made from two-dimensional NMR data taken at a single temperature. The assignment procedure involved acquisition of phase-sensitive double-quantum-filtered correlation spectra, relayed coherence-transfer spectra, total correlation (homonuclear Hartmann-Hahn) spectra, double- and triple-quantum spectra, and nuclear Overhauser effect spectra. The combination of these complementary experiments simplified and accelerated resonance assignment of the peptide. Individual assignments were made at 20 degrees C for all amide and C alpha protons in the peptide, and for all nonlabile side-chain protons on 26 of the 33 amino acid residues in NP-5. Analysis of the proton-proton nuclear Overhauser effect connectivities, the slowly exchanging amide protons, and the proton chemical shifts in NP-5 indicates that the peptide has a stable, ordered structure in solution. These data also indicate that residues 19-29 in NP-5 are involved in an antiparallel beta-sheet that has a hairpin conformation.     

Structure of an isolated gramicidin A double helical species by high-resolution nuclear magnetic resonance.

A conformational species of gramicidin A has been isolated in dioxane by high pressure liquid chromatography and characterized by circular dichroism and two-dimensional proton nuclear magnetic resonance. Double-quantum filtered two-dimensional correlation spectroscopy, two-dimensional homonuclear Hartman Hahn spectroscopy and two-dimensional nuclear Overhauser effect spectra at 500 MHz were used to obtain virtually complete proton assignments and produce 192 distance constraints. Protocols to determine the state of aggregation, monomer-specific assignment of nuclear Overhauser enhancement values, hydrogen bonding pattern and helix handedness are described. A distance geometry/simulated annealing routine was used to generate well-defined backbone and side-chain structures. The species isolated is a right-handed intertwined double helix, with approximately 5.7 residues per turn. Unique values for helical dimensions are also specified.     

Comparison of the solution conformations of human [Zn7]-metallothionein-2 and [Cd7]-metallothionein-2 using nuclear magnetic resonance spectroscopy.

The solution structure of native human [Zn7]-metallothionein-2 has been compared with the previously determined structure of human [Cd7]-metallothionein-2. The comparison was based on complete sequence-specific 1H nuclear magnetic resonance assignments for human [Zn7]-metallothionein-2 obtained using the sequential assignment method. The secondary structure was found to be very similar in the [Zn7]- and [Cd7]- forms of the protein. Only seven amide protons in [Zn7]- metallothionein-2 were found to have exchange rates lower than approximately 0.2 min-1 at pH 7.0 and 10 degrees C, which corresponds closely to the results of amide proton exchange studies with the [Cd7]- form of the protein. Finally, the 1H-1H distance constraints determined from nuclear Overhauser enhancement spectroscopy for human [Zn7]-metallothionein-2 were checked for compatibility with the [Cd7]-metallothionein-2 structure. Overall, although no direct method is available for identifying the metal-polypeptide co-ordinative bonds in the Zn(2+)-containing protein, these measurements provided several independent lines of evidence showing that the [Zn7]- and [Cd7]- forms of human metallothionein-2 have the same molecular architecture.     

1H nuclear magnetic resonance double resonance study of oxytocin in aqueous solution.

Peptide NH resonances in the 250 MHZ 1H nuclear magnetic resonance (NMR) spectrum of oxytocin in H2O were assigned to specific amino acid residues by the "underwater decoupling" technique (i.e., decoupling from corresponding CalphaH resonances, which are buried beneath the intense water peak). These experiments confirm previous assignments of A. I. Brewster an V. J. Hruby ((1973), Proc. Natl. Acad. Sci. U.S.A. 70, 3806) and A. F. Bradbury et al. ((1974), FEBS Lett. 42, 179). Three methods of assigning NH resonances of peptides--solvent titration, underwater decoupling, and isotopic labeling--are compared. As the solvet composition is gradually changed from dimethyl sulfoxide to H2O, oxytocin undergoes a conformational change at 70-90 mol % of H2O. Exposure to solvent of specific hydrogens of oxytocin in H2O was studied by monitoring intensity changes of solute resonances when the solvent peak was saturated. Positive nuclear Overhauser effects (NOE's) of 14 +/- 5 were observed for the Tyr ortho CH and meta CH resonances, respectively. Comparative studies with deamino-oxytocin indicate that these effects result predominantly from intermolecular dipoledipole interaction between aromatic side chain CH protons and protons of the solvent. The NOE's therefore indicate intimate contact between water and the aromatic CH hydrogens of the Tyr side chain. The extent of saturation transferred by proton exchange between water and NH group varies with Ph in a manner which appears to reflect the acid-base catalysis of the protolysis reaction. There is no indication that any NH protons are substantially shiedled from the solvent.     

Sequence-specific 1H NMR assignments and identification of slowly exchanging amide protons in murine epidermal growth factor

Proton nuclear magnetic resonance (1H NMR) assignments for the murine epidermal growth factor (mEGF) in aqueous solution were determined by using two-dimensional NMR at pH 3.1 and 28 degrees C. The assignments are complete for all backbone hydrogen atoms, with the exception of the N-terminal amino group, and for 46 of the 53 side chains. Among the additional seven amino acid residues, three have complete assignments for all but one side-chain proton, and between two and four protons are missing for the remaining four residues. The sequential assignments by nuclear Overhauser effect spectroscopy are consistent with the chemically determined amino acid sequence. The NMR data show that the conformations of both the Tyr3-Pro4 and Cys6-Pro7 peptide bonds are trans in the predominant solution structure. Proton-deuterium exchange rate constants were also measured for 13 slowly exchanging amide protons. The information presented here has been used elsewhere to determine the three-dimensional structure of mEGF in aqueous solution.     

Porcine pancreatic phospholipase A2: sequence-specific 1H and 15N NMR assignments and secondary structure

The solution structure of porcine pancreatic phospholipase A2 (124 residues, 14 kDa) has been studied by two-dimensional homonuclear 1H and two- and three-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Backbone assignments were made for 117 of the 124 amino acids. Short-range nuclear Overhauser effect (NOE) data show three alpha-helices from residues 1-13, 40-58, and 90-109, an antiparallel beta-sheet for residues 74-85, and a small antiparallel beta-sheet between residues 25-26 and 115-116. A 15N-1H heteronuclear multiple-quantum correlation experiment was used to monitor amide proton exchange over a period of 22 h. In total, 61 amide protons showed slow or intermediate exchange, 46 of which are located in the three large helices. Helix 90-109 was found to be considerably more stable than the other helices. For the beta-sheets, four hydrogen bonds could be identified. The secondary structure of porcine PLA in solution, as deduced from NMR, is basically the same as the structure of porcine PLA in the crystalline state. Differences were found in the following regions, however. Residues 1-6 in the first alpha-helix are less structured in solution than in the crystal structure. Whereas in the crystal structure residues 24-29 are involved both in a beta-sheet with residues 115-117 and in a hairpin turn, the expected hydrogen bonds between residues 24-117 and 25-29 do not show slow exchange behavior. This and the absence of several expected NOEs imply that this region has a less well defined structure in solution. Finally, the hydrogen bond between residues 78-81, which is part of a beta-sheet, does not show slow exchange behavior.